968
Anal. Chem. 1981, 53, 968-971
(7) Webb, J. L. “Enzyme and Metabollc Inhlbitors”; Academic Press: New York, 1966; Vol. 3, Chapter 6. (8) Iverson, D. G.; Anderson, M. A.; Holm, T. R.; Stanforth, R. R. Environ. Sci. Technol. 1979. 13. 1491. (9) Johnson, D. L.; Pilson, M. E. A. Anal. Chim. Acta 1972, 58, 289. (10) Henry, F. T.; Thorpe, T. M. Anal. Chem. 1980, 52, 80. (11) Braman, R. S.; Foreback, C. C. Sclence 1973, 182, 1247. (12) Andreae, M. 0. Anal. Chem. 1977, 49, 820. (13) Andreae, M. 0. Deep-sea Res. 1978, 25, 391. (14) Talml, Y.; Bostik, D. T. Anal. Chem. 1975, 47, 2145. (15) Carvalho, M. B.; Hercules, D. M. Anal. Chem. 1978, 50, 2030. (16) Braman, R. S.;Justen, L. L.;Foreback, C. C. Anal. Chem. 1972, 44, 2195. (17) Pierce, F. D.; Brown, H. R. Anal. Chem. 1976, 48, 693.
(18) Smlth, A. E. Analyst (London) 1975, 100. 300. (19) Zatka, V. J. Ana/. Chem. 1978, 50,538. (20) Zief, M.; Mitchell, J. W. “Contamination Control in Trace Element Analvsis:” Wilev: New York. 1976: -. Chanter - -r - 6. (21) Edige;, d. At.-j\bso$t. News/.-1975, 14, 127
RECEIVED for review October 27, 1980. Accepted February 17, 1981. This research was supported in part by NOAA, Office of Sea Grant, through an institutional grant to the University of wisconsin, and by a grant ( N ~D, A ~ ~ C-0046) from the U.S. Army Corps of Engineers.
Determination of Alkaline Earth Metals by Ion-Exchange Chromatography with Spectrophotometric Detection Michlo Zenki Department of Chemistty, Okayama University of Science, 1- 1, Rldai-cho, Okayama-shi, 700, Japan
A liquid chromatographlc-spectrophotometric system has been developed to separate magnesium, calclum, strontlum, and barium. The eluent used is 0.7 M sulfosalicylic acid containing chlorophosfonazo 111 as a color-forming reagent. The separated ions after passing through a catlon-exchange column are detected dlrectly by a spectrophotometric detector. The detection limlt Is 50, 2, 6, and 20 ng for magneslum, calclum, strontium, and barium, respectlveiy. The determination of calclum In natural waters was carried out by this method.
The separation and determination of alkaline earth metals have been of great interest in analytical chemistry, because these metals exist widely in nature and occur together in significant amounts. They seem to play an important role in the field of biology. Although atomic absorption spectrophotometry or flame spectrophotometry has been used for sensitive analysis of alkaline earth metals, it is useful for the individual metal but is not available for their simultaneous and automatic determination. Separations of various metals have been achieved by using ion-exchange chromatography, but most separations are still achieved by analyzing the collected fractions from the column. Recently, high-performance liquid chromatographic techniques (HPLC) for the separation and determination of organic compounds has advanced greatly, but ion-exchange chromatography of metal ions has not. The fact that few detectors are suitable for direct and sensitive detection of metal ions is responsible. Fritz and co-workers (1,Z) reported the ion-exchange separation and determination of calcium and magnesium with spectrophotometric detection. A colorforming reagent and buffer solution were added by pump after column separation of sample ions. The reagent, buffer, and eluate were mixed in a mixing coil (postcolumn reaction). Small et al. (3) employed conductance detection after removal of the eluent by a “stripper” column. Freed ( 4 ) used flame emission for detection of calcium, strontium, and barium. The author has investigated the use of bisazochromotropic acid derivatives as sensitive reagents for several metals ( 5 6 ) . The reagents form water-soluble complexes with alkaline earth
metals and have large molar absorptivities (lo4)over a wide pH range. Although Fritz and co-workers ( I , 2) had previously used these reagents in several postcolumn reaction chromatographic studies, no investigation of their use for the determination of alkaline earth metals has been published. This paper reports the application of these reagents for the HPLC separation of alkaline earth metals with subsequent on-line spectrophotometric determination. The postcolumn reaction was not employed in this work, because controlling pH in the color reaction was very difficult. Thus, the bisazochromotropic acid derivatives are mixed with the eluent prior to being passed thraugh the separation column. The effluent is then introduced directly to a visible spectrophotometric detector. The pump which delivers the reagent and buffer solution, and the mixing tee, chamber, or coil are not necessary.
EXPERIMENTAL SECTION Apparatus. Liquid chromatography was performed with a Yanagimoto L-2OOOL (Yanagimoto Co., Kyoto, Japan) and Rheodyne 7125 sample injector (Rheodyne Inc., Berkeley, CA). The analytical column was a 2.6 nun i.d. X 150 mm length s w e s s steel tube fitted with a water jacket. The temperature used in this work was 25 “C. The detector was a Hitachi Model 200 spectrophotometer (Hitachi Scientific Instruments, Tokyo, Japan). Its sample compartment was altered to accommodate a flowthrough cell having a light path of 8 mm and internal volume of 8 pL. An airtight microsyringe (25 pL) was used to inject the samples. Ion-Exchange&sin, A Hitachi custom cation exchange resin, 2613, was slurry packed into a stainless steel column. The resin consisted of porous polymer particles having sulfonic acid ionic group in a styrene-divinylbenzene matrix. The average particle size was about 17 pm. The resin was conditioned with a sufficient amount of eluent solution before use. Reagents. Unless otherwise noted, all chemicals were analytical reagent grade quality and were used as received. Chlorophosfonazo I11 was obtained from Dojindo Laboratories (Kumamoto, Japan) and used without further purification. Other bisazochromotropic acid derivatives were synthesized and purified in our laboratory (6). Metal ion solutions were prepared by appropriate dilution from 1000 ppm standard solutions supplied by Wako Pure Chemicals Co. (Osaka, Japan). Deionized-distilled water was used throughout. Eluent. For the preparation of eluent solution, 152.6 g of sulfosalicylic acid (Wako Pure Chemicals Co.) and 40 mg of
0003-2700/81/0353-0968$01.25/00 1981 American Chemical Society
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~
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 7, JUNE 1981
Table I. Molar Absorptivities of Bisazochromotropic Acid Derivatives with Alkaline Earth Metal Ions molar absorptivity, L mol-' cm'l X lom4 ___ PH reagents Mg Ca Sr Ba neo-thorin arsenazo I11
0.83 0.3
carboxyarsenazo sulfonazo I11
0.09 0.01
chlorophosf on azo 111
2.66 4.8
0.68 1.6 4.4 0.44 0.01 0.1 3.76 6.4 12.5
carboxynitrazo
\i
# I-
z g10
-
4\ \
0.25 0.72 4.0 0.85 1.07 0.7 4.1
ref 2 8 10 8 8 5 8 9 11
9 5.0-6.8 9.1 7.0 2.4 4.0 7.5 7.0 2.5-4.5
0.14 3.6 0.92 1.12 2.8 4.2 11.0
969
w
\
2
P LLI a
I
I
1.o 1.2 CONCENTRATIONOF SULFOSALICYLIC ACID ( M )
0.6
0.8
I
I
1k
3
7
5
PH
9
Figure 1. Retention time for alkaline earth metals under various sulfosalicylic acid concentrations: 2 X I0.5 M chlorophosfonazo 111; pH 7.5;flow, 1.5 mL/min.
Flgure 2. Relationship between eluent pH and detection response: eluent, 0.7 M sulfosalicyllc acid, 2 X lo-' M chlorophosfonazo 111; flow, 1.5 mL/min; [Mg] 30 pg, [Ca] 5 pg, [Sr] 10 pg, [Ba] 15 pg.
chlorophosfonazoI11 were dissolved in approximately 500 mL of distilled water. An ammonia solution (1-+ 1)was added to adjust the pH of the mixture to 7-8, and the volume was made up to 1 L. The concentration of the resulting solutions were 0.7 M for sulfosalicylic acid and 5 X 10" M for chlorophosfonazo 111.
Table 11. Comparison of Bisazochromotropic Acid Derivatives under the Chromatographic Condition" absorbance X l o 3 length, wave-
RESULTS AND DISCUSSION Elution. Inorganic acids, salts, and complexing agents are common eluents for the separation of alkaline earth metals (7). Inorganic acids were not considered because the column was stainless steel. From 1 X to 1 M solutions of the following salts or complexing agents were checked for separation of alkaline earth metal ions: potassium chloride, sodium acetate, ammonium acetate, citrate, tartrate, glycine, salicylic acid, sulfosalicylic acid, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid, tram-1,2-cyclohexanediamineN,N,N',N'-tetraacetic acid (CyDTA), ethylenediamine, iminodiacetic acid, lactic acid, DL-malic acid, acrylic acid, a-hydroxyisolactic acid, glyceric acid, benzilic acid, mandelic acid, and hydrocinnamic acid. Of those, ammonium acetate, tartrate, sulfosalicylic acid, EDTA, CyDTA, lactic acid, aL-malic acid, a-hydroxyisolactic acid, acrylic acid, and glyceric acid were found to be better for the separation of alkaline earth metals from each other. Retention times were within 30 min. Sulfosalicylic acid was found to be the best when considering retention time and detection sensitivity and was used throughout this work. The relationship between the concentration of s&osalicylic acid and the retention time of alkaline earth metals is shown in Figure 1. The increase of the concentration of sulfosalicylic acid results in a faster retention time but a lowering of the sensitivity. The concentration of sulfosalicylic acid was decided to be 0.7 M by considering the retention time of barium within 15 min. Various eluents in the pH range from 2 to 9 were prepared, and the effect of pH on the retention time or detection intensity was investigated. The retention time does not change
reagentb
hfg
Ca
Sr
neo-thorin arsenazo I11 car boxy arsenaz o sulfonazo I11 chlorophosfonazo I11 carboxynitrazo
17 7 4
1 15 2
6 1
21
56
35
Ba
nm
3 3 1 24
556 655 655 645 679 710
a The experimental conditions used in this study are similar to those given in Figure 2. Concentration of each reagent is 2 x &I.
above pH 4, but the detection intensity is strongly affected by the pH of the eluents (Figure 2). The sensitivity of bisazochromotropic acid derivatives for the metal ions is reduced by the masking effect of sulfosalicylicacid, especially at about pH 5. Although the color reactions of bisazochromotropic acid derivatives with metal ions ordiaarily occur in acidic solution where the maximum sensitivity is obtained (a),in this system it was preferable to carry out the measurement in a neutral or basic solution. The best separation and sensitivity of alkaline earth metals are obtained in the pH range from 6 to 8. Bisazochromotropic Acid Derivatives. Of the bisazochromotropic acid derivatives, neo-thorin (arsenazo I, monoazo derivative), arsenaho 111, carboxyarsenazo, sulfonazo 111, chlorophosfonazo 111,and carboxynitrazo were chosen because they had high molar absorptivities for alkaline earth metals (Table I). Table I1 shows the comparison of these reagents in the chromatographic condition at pH 7.5. Carboxynitrazo which shows a great molar absorptivity (105) cannot be used because of the formation of the precipitate
970
ANALYTICAL CHEMISTRY, VOL. 53, NO. 7,JUNE 1981
Table III. Permissible Amounts of Foreign Ions for Determination of Calcium" ions 2000 56 10 5 0.1 0.01
alkali metal ions BeZ+,Fe3+,A13+,MnZt, Cr3+ Sn2+,Se4+,V5+,Hgz+,Te4+ Cu*+,Ti4+,Si4+,Sb3+,PtZ+,PdZ+,Au' Ni2+
rare earth metal ions
a Calcium taken; 0.1 fig. tested.
Maximum concentration
Table IV. Determination of Calcium in Natural Waters I
O1
I
1 2 CHLOROPHOSFONAZO ( ~x
m
I
d)
Flgure 3. Effect of concentration of chlorophosfonazo 111: Eluent, 0.7 M sulfosalicylic acid; pH 7.5; flow, 1.5 mL/min; [Mg] 4.9 pg, [Ca] 0.4 pg, [Srl 1.2 pg, [Ba] 1.9 pg. 0.0 10
t
0
I
I
I
i
I
Ca
3
6 9 12 RETENTION TIME (rnin)
15
Flgure 4. Separation of alkaline earth metals: eluent, 0.7 M sulfoM chlorophosfonazo 111; pH 7.5; flow 1.5 salicylic acid, 5 X mL/min; [Mg] 1.0 pg, [Ca] 0.08 pg, [Sr] 0.24 pg, [Ba] 0.38 pg. in an acidic solution, and it does not react with the alkaline earth metals in a neutral or basic solution. Neo-thorin, one of the monoazochromotropic acid derivatives, seems to be favorable only for the determination of magnesium. Chlorophosfonazo I11 is most sensitive for all alkaline earth metals and was used in further investigations. The appropriate amount of chlorophosfonazo I11 in this system was examined by varying the concentration of chlorophosfonazo I11 from 1 X to 2 X lo4 M. The measurement was run at 679 nm. Figure 3 shows the relatidqship between the concentration of chlorophosfonazo I11 and the detector response. The sensitivity increased almost linearly with increasing the concentration of chlorophosfonazo 111. Since the background absorbance of the reagent is a major factor on the detection sensitivity, the concentration of the reagent should be kept as small as possible, meanwhile it is necessary for a sufficient amount of the reagent to react with alkaline earth metal ions. Therefore, the optimum reagent concentration was determined to be 5 X M for chlorophosfonazo 111. The relationship between the concentration of chlorophosfonazo I11 and the retention time of alkaline earth metals was also investigated. The retention time of calcium, strontium, and barium was accelerated slowly with the increase of chlorophosfonazo I11 concentration. It is evident that chlorophosfonazo I11 has an eluent ability, whereas this phenomenon is not observed with the other reagents such as arsenazo 111. The reason and the mechanism are attributable to the characteristic of phosphonic acid group in the chlorophosfonazo I11 molecule, and it implies the formation
sample a
this method,b PPm River Water
Yoshii River, Okayama Prefecture, Japan Asahi River, Okayama Prefecture, Japan Takahashi River, Okayama Prefecture, Japan
7.70
atomic absorption method, ppm
0.05
7.60
6.75 i 0.02
6.75
+_
13.4 i 0.2
City Water 6.70 ? 0.04 Okayama City, Okayama Prefecture, Japan Kurashiki City, 13.2 i. 0.2 Okayama Prefecture, Japan a All samples collected on July 22, 1980. value of three determinations.
13.2
6.75 12.9
The mean
of a ternary complex, sulfosalicylic acid-metal ion-chlorophosfonazo 111. Separations. Figure 4 shows a chromatogram of alkaline earth metals obtained in this work. The order of elution of ions is in accord with a pfevious work (7). Excellent separatiori wds achieved within 15 min; t h b , the stepwise elution or gradient elution is not necessary (2). Calibration plots were constructed for these ions, and the close linear dependencies were obtained between the peak height and the amount of the ions over a wide range of concentration. This method is very sensitive for calcium, strontium, and barium but less sensitive for magnesium. The detection limits are 50,2,6, and 20 ng for magnesium, calcium, strontium, and barium, respectively. The relative standard deviation in the determination of calcium was 1.16% for 10 replicate runs. Interferences from several other metal ions were also investigated. Table I11 shows the tolerance limit of the other metal ions which cause 5% error on the determination of calcium ion. Most metal ions can be eluted prior to or overlapping with magnesium peak and thus do not affect the determination of calcium, strontium, and barium. Although nickel and rare earth metal ions also elute prior to calcium peak, they interfered with the calcium determination because they formed a stable complex with chlorophosfonazo I11 and gave a large peak. Application. The method mentioned above is sensitive enough to permit the direct determination of calcium in natural water samples. Table IV shows the results of analysis of calcium in local river waters and city waters by this method. A 25-pL sample was directly injected into the column by an
Anal. Chem. 1981, 53, 971-975
airtight syringe without any prior sample treatment. Results from the chromatographic method are compared to atomic absorption results obtained with an air-acetylene flame. The results from the two techniques are fairly coincident with each other within 3% of relative errors.
ACKNOWLEDGMENT The author is indebted to Professor Kyoji T6ei of Okayama University and to Professor Toyokichi Kitagawa of Osaka City University for valuable advice and discussion.
LITERATURE CITED (1) Fritz, J. S.; Story, J. N. Anal. Chem. 1974, 46, 825-829. (2) Argueilo, M. D.; Fritz, J. S. Anal. Chem. 1977, 49, 1595-1598.
971
(3) Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, 1801-1809. (4) Freed, D. J. Anal. Chem. 1975, 47, 186-187. (5) Zenki, M. Anal. Chlm. Acta 1978, 83, 267-274. (6) Zenki, M. Anal. Chim. Acta 1977, 93, 323-326. (7) Samuelson, 0. “Ion Exchange Separation in Analytical Chemistry”; Wiley: New York, 1963; Chapter 15. (8) Flaschka, H. A.; Barnard, A. J., Jr. “Chelates In Analytical Chemistry”; Marcel Dekker: New York, 1969; Vol. 2, p 1. (9) Ferguson, J. W.; Richard, J. J.; O’iaughlin, J. W.; Banks, C. V. Anal. Chem. 1984, 36, 796-799. (IO) Michayiova, V.; Kouleva, N. Talanta 1974, 21, 523-532. (11) Sawin, S. 8.; Petrova, T. V. Zh. Anal. Khlm. 1989, 24, 177-185.
RECEIVED for review November 21, 1980. Accepted March 2, 1981.
Liquid Chromatography with Real-Time Video Fluorometric Monitoring of Effluents L. W.
Hershberger, 9. B. Callis, and G. D. Christian*
Department of Chemistry BG- 10, University of Washington, Seattle, Washington 98 195
Real-time fluorescence monltorlng at multiple wavelengths of excitation and emission is performed by Interfaclng a highperformance liquid chromatograph to the video fluorometer using a laminar flow cell to mlnimlre dead volume and scattered Ilght. A total fluorescence chromatogram, as well as two selected excitation-emlssion wavelength chromatograms, are displayed in real time. For perylene, the linear dynamic range covers at least 2 orders of magnitude wlth a detectlon limit of 1 ng. Selected fluorescence monitoring Is capable of spectrally separating and quantifying benzo[a]pyrene and benro[elpyrene whose chromatographic retentlon profiles overlap slgniflcantly. Flnally, a complex mixture, shale oil, is analyzed for benzolalpyrene.
The combination of a separation technique with a molecular fingerprinting technique offers the most powerful means of analyzing the complex materials with which today’s analytical chemist must be concerned ( I ) . Obviously, the combination against which all others must be compared is gas chromatography/mass spectrometry (GC/MS). In cases where the components to be analyzed lack sufficient volatility for gas chromatography, liquid chromatography combined with a fingerprinting technique such as mass spectrometry or rapid-scanning UV-Vis absorption spectrometry offers a path to analysis. Very recently, Winefordner and co-workers (2)and Talmi and co-workers (3, 4 ) have evaluated the efficacy of rapid scanning fluorescence detection of high-performance liquid chromatography (HPLC) effluents. Winefordner (2) showed with simulated chromatography data that multichannel imaging detectors based upon the SIT vidicon provided sufficient sensitivity for trace analysis. Talmi and coworkers (3,4)showed that an HPLC-multichannel fluorometer could easily be assembled from commercial parts, and their preliminary work showed that a qualitative analysis of the aromatic hydrocarbon fraction was quite feasible. Independent of these and other efforts (5-10) to use multichannel imagers as fluorescence detectors, we developed the video fluorometer (11). This instrument is unique because it uses a novel irradiation geometry to obtain excitation and emission
spectra simultaneously. Recent studies have demonstrated that the video fluorometer is capable of determining selected components in a 12-component mixture, even under circumstances where spectral overlap is severe and intensities vary over a couple of orders of magnitude (12-14). Thus, the combination of the video fluorometer and HPLC seems especially appealing because the resulting system would use three dimensions to separate mixtures (excitation wavelength, emission wavelength, and chromatographic retention time). In this exploratory paper, we report on the potential of the liquid chromatograph/video fluorometer (LC/VF) for analysis of complex mixtures. Our capability to display the data as EEMs and as selected excitation-emission wavelength chromatograms is shown to be extremely helpful to the operator of the instrument.
EXPERIMENTAL SECTION Flow Cell. A modified version (Figure 1) of the sheath flow cell previously described (15)was used as the flow cuvette in the video fluorometer. In the modified version, the quartz windows (G) and the laminar flow path were increased in length to 17 mm to permit placement in the cuvette position of the video fluorometer without modification to the optical arrangement. Standard HPLC tubing and fittings were used to simplify connections to the flow cell. The sheath alignment (A) and exit bores (I) were 1/8 in. in diameter. The two 1/32 in. diameter sheath inlets (B) were drilled into the top of the sample alignment bore. Connections to the sheath inlets were made using a ‘/I6 in. Swagelok male plug (C) soldered with the cap end to each sheath inlet and a hole drilled through the cap. The chromatographic effluent was introduced into the sample alignment bore from a piece of 0.02 in. i.d. and ’/I6 in. 0.d. stainless steel tubing (D). The sample inlet tube was aligned and held in the center of the inlet bore by a ‘Ile in. Altex male fitting (E) and a Teflon ferrule (F). The outlet from the in. Swagelok male plug (H) attached flow cell used a drilled by the same method as the sheath inlets. Each of the four l/s in. thick quartz windows (G)was held and sealed against a Teflon in. stainless steel plate (J)using screws. The gasket (K) by a four steel plates contained openings for the windows. Under the laminar flow conditions, the diameter of the sheath and sample stream was that of the entrance and exit (I) ports, Le., 1/8 in. The flowing stream was surrounded by stagnant sheath solvent that filled the void between the stream and the windows.
0003-2700/81/0353-C971$01.25/00 1981 American Chemical Society
